The analysis of circulating nucleic acids has revealed applications in the non-invasive diagnosis, monitoring, and prognostication of many clinical conditions. For example, for prenatal applications, circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of sequence differences (e.g., single-nucleotide polymorphisms, or SNPs) between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical methods.
Current techniques to detect sequence differences in a DNA sample include allele-specific PCR, restriction digest and Southern blot hybridization, restriction endonuclease-mediated selective-PCR (REMS-PCR), and competitive PCR methods involving the use of fluorescent detection probes. Currently available techniques present several disadvantages. For allele-specific PCR, it is often difficult to design assays with a high degree of allele specificity (Nasis et al. Clin Chem. 2004 April; 50(4):694-701). Restriction digest/Southern blot methods require higher amounts of DNA template than the method provided herein, and lack the sensitivity to detect polymorphic sequences comprising a low relative proportion of total DNA. Restriction endonuclease-mediated selective-PCR (REMS-PCR) has the drawback of requiring a thermostable restriction enzyme that cleaves the wild-type allele. REMS-PCR is described in U.S. Pat. No. 6,261,768, which is hereby incorporated by reference. Use of the technique may not always be possible, and this requirement limits the general utility of the REMS-PCR approach. Competitive PCR lacks the sensitivity to detect polymorphic sequences comprising a low relative proportion (<5%) of total DNA. Competitive PCR with allele-specific fluorescent probes lacks the ability to multiplex assays higher than 2-3 assays in a single tube format. In addition, similar methods utilizing methylation differences between DNA species (for example, US Patent Application Publication No. 20070059707, entitled, “Methods for prenatal diagnosis of chromosomal abnormalities”, which is hereby incorporated by reference) are not effective at low copy numbers of genomic DNA.